Biosynthesis of Rhamnosylated Anthraquinones in Escherichia coli

Rhamnose is a naturally occurring deoxysugar present as a glycogenic component of plant and microbial natural products. A recombinant mutant Escherichia coli strain was developed by overexpressing genes involved in the TDP-L-rhamnose biosynthesis pathway of different bacterial strains and Saccharothrix espanaensis rhamnosyl transferase to conjugate intrinsic cytosolic TDP-L-rhamnose with anthraquinones supplemented exogenously. Among the five anthraquinones (alizarin, emodin, chrysazin, anthrarufin, and quinizarin) tested, quinizarin was biotransformed into a rhamoside derivative with the highest conversion ratio by whole cells of engineered E. coli. The quinizarin glycoside was identified by various chromatographic and spectroscopic analyses. The anti-proliferative property of the newly synthesized rhamnoside, quinizarin-4-O-α-L-rhamnoside, was assayed in various cancer cells.

color gasoline and heating oil; it also acts as an intermediate for the synthesis of indanthrene-and alizarin-derived dyes [2]. Further, anthraquinone glycosides exhibit stronger activity than free aglycones [19].
First, we employed a versatile post-biosynthesis modifying enzyme, O-rhamnosyltransferase (7665), derived from S. espanaensis for the biosynthesis of anthraquinone rhamnosides in the engineered E. coli mutant strain overexpressing genes for TDP-L-rhamnose. Five different anthraquinones were added for biotransformation into respective O-rhamnosides. After 20 h of isopropyl β-D-1-thiogalactopyranoside (IPTG)-induced culture, we supplemented anthraquinone exogenously at a final concentration of 0.2 mM. The biotransformation reaction was continued for the next 28 h at 20°C, followed by extraction using a double volume of ethyl acetate and analysis via high-performance liquid chromatography (HPLC-PDA).
The HPLC-PDA analysis of each sample yielded product peaks at shorter retention times (t R ) than the substrate peak in each reaction mixture, as expected. New peaks appearing at t R ~ 19.6 min in alizarin (t  The genes (pgi, zwf, and galU) were knocked out of the genome. The dTDP-L-rhamnose was generated in the cytosol of engineered E. coli by overexpressing the respective genes in the sugar pathway. Rhamnosyl transferase (7665) from S. espanaensis was used for the conjugation of sugar to the exogenously supplemented anthraquinones (emodin, chrysazin, alizarin, anthrarufin, and quinizarin).  (Fig. S2). The biotransformation reaction analysis by HPLC-PDA and ESI/MS revealed that the engineered strain converted all exogenously supplemented substrates to products. The conversion percentage of emodin, chrysazin, quinizarin, anthrarufin and alizarin were 2.4%, 2.5%, 17%, 10.7%, and 3%, respectively. Based on the highest conversion, a further study of quinizarin alone was carried out.
We increased the bioconversion of quinizarin via supplementation of different concentrations (0%, 2%, 4%, 6%, and 8%) of glucose in cultures grown under identical conditions during biotransformation. The change in conversion percentage of quinizarin to product was monitored at different time intervals (from 0 to 60 h). The result showed that supplementation of 2% additional glucose improved the conversion from 22% (36 h, without additional glucose) to 75% (48 h) (Figs. S3 and S4). The addition of glucose facilitated cell growth and product yield.
The product was purified by using prep-HPLC and then subjected to nuclear magnetic resonance (NMR) analyses. While comparing the 1 H NMR spectra of standard quinizarin and the reaction product, signals from the parent compound containing 2-hydroxyl groups in the symmetrical position were detected at δ 12.71 (1H, s) while in the reaction compound hydroxyl group, the signals were detected at δ 12.88 (1H, s) ( Table 1, Figs. S5a and S5b). The anomeric proton (1'-H) was consistent with δ 5.48 (d, J = 1.7 Hz, 1H); however, the anomeric proton coupling constant (J = 1.7 Hz) confirmed that the conjugation of rhamnose moiety was in α-configuration. In addition, based on the 1 3 C NMR analysis of the reaction product, the new peak appeared at δ 100.01 ppm for anomeric carbon and other carbon peaks between 70 and 80 ppm along with a CH coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) spectroscopy. The result supported the correlation between the observed anomeric carbon and anomeric proton revealed by HSQC (Fig. S7). Similarly, the carbon C-4 of the quinizarin signal appearing at δ 150.48 ppm showed a direct correlation with the observed anomeric proton at δ 5.48 ppm in HMBC (Fig. S8). Based on these results, the product was established as quinizarin 4- Previous studies showed that the anticancer effects of anthraquinones were associated with the suppression of cancer cell proliferation [25]. We thus evaluated the effects of quinizarin and its derivative on the proliferation of A375SM melanoma, AGS gastric cancer, MCF-7 breast cancer, and U87MG brain cancer. The inhibitory effect of quinizarin rhamnoside was greater than that of aglycone in all cancer cell lines tested. This result showed that approximately 70% of AGS gastric cancer cells failed to grow in the presence of 50 μM concentration of quinizarin rhamnoside while the suppression of cell growth was only 20% under the same concentration of quinizarin. Although subtle growth reduction was observed with rhamnoside derivative, the decrease in cell proliferation of MCF-7 breast cancer cells and U87MG brain cancer was not significantly different with quinizarin and its rhamnoside derivative (Fig. 3).
Chemical synthesis of anthraquinone glycosides requires multiple steps, uses hazardous chemicals, and is therefore an environmentally unfriendly approach [26]. Moreover, production of anthraquinone rhamnosides in practical quantities from plant sources has been tedious and impractical as biosynthesis in large quantity from these sources is difficult to achieve while purification and extraction are more challenging because of the presence of a large number of other metabolites [27]. Therefore, regiospecific biosynthesis using engineered recombinant microbial cells is superior in terms of sustainability while being eco-friendly and enabling easy fermentation and scale-up for industrial biosynthesis [28]. Thus, this study provides a broad overview of the modification of anthraquinones by rhamnosylation using an engineered E. coli strain in a sustainable way. The antiproliferative activities